Synaptic transmission depends on the efficient packaging of neurotransmitters into specialized secretory vesicles. For monoamines that are synthesized in the cytoplasm, this package requires active transport across the vesicle membrane. Drugs that interfere with vesicular amine transport have profound psychotropic effects, indicating the importance of this activity in psychiatric diseases. Interestingly, monoamines are stored in two distinct secretory vesicles: synaptic vesicles (SVs) and large dense core vesicles (LDCVs). SVs mediate fast synaptic transmission, while LDCVs have a neuromodulatory role. Whereas the specific transport activity defines the content of secretory vesicles, the localization of the transport protein on distinct vesicles will determine the mode and site of monoamine release. The long-term objective of this proposal is to understand how changes in neurotransmitter storage and release influence synaptic transmission, and hence behavior. The strategy is to study the sorting of the transport proteins that package monoamines into distinct secretory vesicles. Using PC 12 cells as a model system, we have found that vesicular monoamine transporters (VMATs) preferentially localize to LDCVs whereas the close related vesicular acetylcholine transporter (VAChT), preferentially to SVs. We will focus on neuronal VMAT2 in this proposal to explore the mechanisms involved in the sorting of VMAT2 to LDCVs with the following Specific Aims: 1) To examine the routes by which VMAT2 sorts to secretory vesicles. 2) To identify the sorting sequences that target VMAT2 to LDCVs. 3) To identify interacting proteins that regulate VMAT2 sorting. The proposed studies will identify the routes, sorting sequences, and the regulatory proteins involved in the sorting of VMAT2 to secretory vesicles in neuroendocrine cells and neurons. This information will facilitate our understanding of how the monoaminergic neural transmission is regulated at the level of storage and release. This work will also allow us in the future to genetically alter the localization of VMAT2 in vivo and determine the biological significance of monoamine release from LDCVs as well as SVs, which could lead to better understanding of the functions regulated by aminergic pathways, such as those underlying drug seeking and psychotic behaviors.